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We report that ribavirin exerts an inhibitory and mutagenic activity on SARS-CoV-2-infecting Vero cells, with a therapeutic index higher than 10. Deep sequencing analysis of the mutant spectrum of SARS-CoV-2 replicating in the absence or presence of ribavirin indicated an increase in the number of mutations, but not in deletions, and modification of diversity indices, expected from a mutagenic activity. Notably, the major mutation types enhanced by replication in the presence of ribavirin were A→G and U→C transitions, a pattern which is opposite to the dominance of G→A and C→U transitions previously described for most RNA viruses. Implications of the inhibitory activity of ribavirin, and the atypical mutational bias produced on SARS-CoV-2, for the search for synergistic anti-COVID-19 lethal mutagen combinations are discussed.

RNA viruses replicate as complex mutant spectra termed viral quasispecies. The frequency of each individual genome in a mutant spectrum depends on its rate of generation and its relative fitness in the replicating population ensemble. The advent of deep sequencing methodologies allows for the first-time quantification of haplotype abundances within mutant spectra. There is no information on the haplotype profile of the resident genomes and how the landscape evolves when a virus replicates in a controlled cell culture environment. Here, we report the construction of intramutant spectrum haplotype landscapes of three amplicons of the NS5A-NS5B coding region of hepatitis C virus (HCV). Two-dimensional (2D) neural networks were constructed for 44 related HCV populations derived from a common clonal ancestor that was passaged up to 210 times in human hepatoma Huh-7.5 cells in the absence of external selective pressures. The haplotype profiles consisted of an extended dense basal platform, from which a lower number of protruding higher peaks emerged. As HCV increased its adaptation to the cells, the number of haplotype peaks within each mutant spectrum expanded, and their distribution shifted in the 2D network. The results show that extensive HCV replication in a monotonous cell culture environment does not limit HCV exploration of sequence space through haplotype peak movements. The landscapes reflect dynamic variation in the intramutant spectrum haplotype profile and may serve as a reference to interpret the modifications produced by external selective pressures or to compare with the landscapes of mutant spectra in complex in vivo environments. IMPORTANCE The study provides for the first time the haplotype profile and its variation in the course of virus adaptation to a cell culture environment in the absence of external selective constraints. The deep sequencing-based self-organized maps document a two-layer haplotype distribution with an ample basal platform and a lower number of protruding peaks. The results suggest an inferred intramutant spectrum fitness landscape structure that offers potential benefits for virus resilience to mutational inputs.

Selection of viral mutants resistant to compounds used in therapy is a major determinant of treatment failure, a problem akin to antibiotic resistance in bacteria. In this scenario, mutagenic base and nucleoside analogs have entered the picture because they increase the mutation rate of viral populations to levels incompatible with their survival. This antiviral strategy is termed lethal mutagenesis. It has found a major impulse with the observation that some antiviral agents, which initially were considered only inhibitors of virus multiplication, may in effect exert part of their antiviral activity through mutagenesis. Here, we review the conceptual basis of lethal mutagenesis, the evidence of virus extinction through mutagenic nucleotide analogs and prospects for application in antiviral designs., European Regional Development Fund (ERDF). CIBERehd (Centro de Investigacion en Red de Enfermedades Hepáticas y Digestivas) is funded ´ by Instituto de Salud Carlos III. T BFU-2011-23604, SAF2014-52400-R from Ministerio de Economía y Competitividad, SAF2017-87846-R, BFU2017-91384-EXP, PI18/00210 from Ministerio de Ciencia, Innovacion y Universidades, and S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER) and P2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER) (http://platesa-es.weebly.com). The work in Barcelona was also funded by Instituto de Salud Carlos III; by grants PI15/00829 and PI16/00337 cofinanced by the ERDF; and by CDTI (Centro para el Desarrollo Tecnologico Industrial), ´Spanish Ministry of Economics and Competitiveness (MINECO), IDI-20151125. Institutional grants from the Fundacion Ramón Areces and Banco Santander to the CBMSO

Replication of RNA viruses is characterized by exploration of sequence space which facilitates their adaptation to changing environments. It is generally accepted that such exploration takes place mainly in response to positive selection, and that further diversification is boosted by modifications of virus population size, particularly bottleneck events. Our recent results with hepatitis C virus (HCV) have shown that the expansion in sequence space of a viral clone continues despite prolonged replication in a stable cell culture environment. Diagnosis of the expansion was based on the quantification of diversity indices, the occurrence of intra-population mutational waves (variations in mutant frequencies), and greater individual residue variations in mutant spectra than those anticipated from sequence alignments in data banks. In the present report, we review our previous results, and show additionally that mutational waves in amplicons from the NS5A-NS5B-coding region are equally prominent during HCV passage in the absence or presence of the mutagenic nucleotide analogues favipiravir or ribavirin. In addition, by extending our previous analysis to amplicons of the NS3- and NS5A-coding region, we provide further evidence of the incongruence between amino acid conservation scores in mutant spectra from infected patients and in the Los Alamos National Laboratory HCV data banks. We hypothesize that these observations have as a common origin a permanent state of HCV population disequilibrium even upon extensive viral replication in the absence of external selective constraints or changes in population size. Such a persistent disequilibrium¿revealed by the changing composition of the mutant spectrum¿may facilitate finding alternative mutational pathways for HCV antiviral resistance. The possible significance of our model for other genetically variable viruses is discussed., The work at CBMSO was supported by grants SAF2014-52400-R from Ministerio de Economía y Competitividad (MINECO), SAF2017-87846-R and BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 from Instituto de Salud Carlos III, S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER), and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). The work in Barcelona was supported by Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF) Grant No. PI19/00301 and by the Centro para el Desarrollo Tecnológico Industrial (CDTI) from the MICIU, Grant No. IDI-20200297. Work at CAB was supported by MINECO grant BIO2016-79618R and PID2019-104903RB-I00 (funded by the EU under the FEDER program) and by the Spanish State research agency (AEI) through project number MDM-2017-0737 Unidad de Excelencia “María de Maeztu”-Centro de Astrobiología (CSIC-INTA). C.G.-C. is supported by predoctoral contract PRE2018-083422 from MCIU. B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo), cofinanced by Fondo Social Europeo (FSE).

Despite the high virological response rates achieved with current directly-acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. Identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultra-deep sequencing (UDS) methods for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV from 220 patients who failed therapy, which are not bona fide resistance-associated substitutions (RAS). They were present frequently in basal and post-treatment virus of patients who failed therapy to different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Coherently, their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. Also, they have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value of treatment response are discussed.

In the course of experiments aimed at deciphering the inhibition mechanism of mycophenolic acid and ribavirin in hepatitis C virus (HCV) infection, we observed an inhibitory effect of the nucleoside guanosine (Gua). Here, we report that Gua and not the other standard nucleosides inhibits HCV replication in human hepatoma cells. Gua did not directly inhibit the in vitro polymerase activity of NS5B, but it modified the intracellular levels of nucleoside di- and tri-phosphate (NDPs and NTPs), leading to deficient HCV RNA replication and reduction of infectious progeny virus production. Changes in the concentrations of NTP or NDP modified NS5B RNA polymerase activity in vitro, in particular de novo RNA synthesis and template switching. Furthermore, the Gua-mediated changes were associated with a significant increase in the number of indels in viral RNA, which may account for the reduction of the specific infectivity of the viral progeny, suggesting the presence of defective genomes. Thus, a proper NTP:NDP balance appears to be critical to ensure HCV polymerase fidelity and minimal production of defective genomes., C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121) cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. The work at CBMSO was supported by grants SAF2014-52400-R from Ministerio de Economía y Competitividad (MINECO), SAF2017-87846-R, BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MICIU), PI18/00210 from Instituto de Salud Carlos III, S2013/ABI-2906, (PLATESA from Comunidad de Madrid/FEDER) and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). The team at CBMSO belongs to the Global Virus Network (GVN). The work in Albacete was supported by grants SAF2016-80451-P, EQC2018-004420-P, and EQC2018-004631-P from MICIU, and by Plan Propio of the University of Castilla-La Mancha. The work in Malaga was supported by Plan Propio of the University of Málaga. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged.

Despite the high virological response rates achieved with current directly acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. The identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultradeep sequencing (UDS) methods, for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide resistance-associated substitutions (RAS), from 220 patients who failed therapy. They were present frequently in basal and posttreatment virus of patients who failed different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. They also have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value for treatment response, are discussed., The work at CBMSO was supported by grants SAF2014-52400-R from Ministerio de Economía y Competitividad (MINECO), SAF2017-87846-R and BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MICIU), PI18/00210 from Instituto de Salud Carlos III, S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER), and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121 and CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). The work in Barcelona was supported by Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF) grant number PI19/00301 and by the Centro para el Desarrollo Tecnológico Industrial (CDTI) from the MICIU, grant number IDI-20151125. Work at CAB was supported by MINECO grant BIO2016-79618R and PID2019-104903RB-I00 (funded by the EU under the FEDER program) and by the Spanish State research agency (AEI) through project number MDM-2017-0737 Unidad de Excelencia “María de Maeztu”-Centro de Astrobiología (CSIC-INTA). Work at IBMB was supported by MICIN grant BIO2017-83906-P (funded by the EU under the FEDER program). C.G.-C. is supported by predoctoral contract PRE2018-083422 from MICIU. B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo), cofinanced by Fondo Social Europeo (FSE).

Previous studies documented that long-term hepatitis C virus (HCV) replication in human hepatoma Huh-7.5 cells resulted in viral fitness gain, expansion of the mutant spectrum, and several phenotypic alterations. In the present work, we show that mutational waves (changes in frequency of individual mutations) occurred continuously and became more prominent as the virus gained fitness. They were accompanied by an increasing proportion of heterogeneous genomic sites that affected 1 position in the initial HCV population and 19 and 69 positions at passages 100 and 200, respectively. Analysis of biological clones of HCV showed that these dynamic events affected infectious genomes, since part of the fluctuating mutations became incorporated into viable genomes. While 17 mutations were scored in 3 biological clones isolated from the initial population, the number reached 72 in 3 biological clones from the population at passage 200. Biological clones differed in their responses to antiviral inhibitors, indicating a phenotypic impact of viral dynamics. Thus, HCV adaptation to a specific constant environment (cell culture without external influences) broadens the mutant repertoire and does not focus the population toward a limited number of dominant genomes. A retrospective examination of mutant spectra of foot-and-mouth disease virus passaged in cell cultures suggests a parallel behavior here described for HCV. We propose that virus diversification in a constant environment has its basis in the availability of multiple alternative mutational pathways for fitness gain. This mechanism of broad diversification should also apply to other replicative systems characterized by high mutation rates and large population sizes. IMPORTANCE The study shows that extensive replication of an RNA virus in a constant biological environment does not limit exploration of sequence space and adaptive options. There was no convergence toward a restricted set of adapted genomes. Mutational waves and mutant spectrum broadening affected infectious genomes. Therefore, profound modifications of mutant spectrum composition and consensus sequence diversification are not exclusively dependent on environmental alterations or the intervention of population bottlenecks., The work at CBMSO was supported by grants SAF2014-52400-R from Ministerio de Economía y Competitividad (MINECO); SAF2017-87846-R and BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MICIU); and S2013/ABI-2906 (PLATESA from Comunidad de Madrid [CAM]/FEDER), S2018/BAA-4370 (PLATESA2 from CAM/FEDER), and PI18/00210 from Instituto de Salud Carlos III. C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121), cofinanced by the European Regional Development Fund (ERDF). CIBERehd is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). The work in Barcelona was supported by Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF) (grant numbers PI16/00337) and by the Centro para el Desarrollo Tecnológico Industrial (CDTI) from the Spanish Ministry of Economy and Business (grant number IDI-20151125). Work at CAB was supported by MINECO grant BIO2016-79618-R (funded by the European Union under the FEDER program) and by the Spanish State Research Agency (AEI) through project number MDM-2017-0737 Unidad de Excelencia “María de Maeztu”-Centro de Astrobiologia (CSIC-INTA).

[Background] Despite the high sustained virological response rates achieved with current directly-acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 5–10% of treated patients do not respond to current antiviral therapies, and basal resistance to DAAs is increasingly detected among treatment-naïve infected individuals. Identification of amino acid substitutions (including those in minority variants) associated with treatment failure requires analytical designs that take into account the high diversification of HCV in more than 86 subtypes according to the ICTV website (June 2017)., [Methods] The methodology has involved five sequential steps: (i) to design 280 oligonucleotide primers (some including a maximum of three degenerate positions), and of which 120 were tested to amplify NS3, NS5A-, and NS5B-coding regions in a subtype-specific manner, (ii) to define a reference sequence for each subtype, (iii) to perform experimental controls to define a cut-off value for detection of minority amino acids, (iv) to establish bioinformatics’ tools to quantify amino acid replacements, and (v) to validate the procedure with patient samples., [Results] A robust ultra-deep sequencing procedure to analyze HCV circulating in serum samples from patients infected with virus that belongs to the ten most prevalent subtypes worldwide: 1a, 1b, 2a, 2b, 2c, 2j, 3a, 4d, 4e, 4f has been developed. Oligonucleotide primers are subtype-specific. A cut-off value of 1% mutant frequency has been established for individual mutations and haplotypes., [Conclusion] The methodological pipeline described here is adequate to characterize in-depth mutant spectra of HCV populations, and it provides a tool to understand HCV diversification and treatment failures. The pipeline can be periodically extended in the event of HCV diversification into new genotypes or subtypes, and provides a framework applicable to other RNA viral pathogens, with potential to couple detection of drug-resistant mutations with treatment planning., This study was supported by the Spanish Health Ministry (Plan Estratégico Nacional contra la Hepatitis C). The work in Barcelona was also funded by Instituto de Salud Carlos III; by grants PI13/00456, PI15/00829, and PI16/ 00337 cofinanced by the European Regional Development Fund (ERDF); and by CDTI (Centro para el Desarrollo Tecnológico Industrial), Spanish Ministry of Economics and Competitiveness (MINECO), IDI-20151125. C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/ 00121) cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. The work in Madrid was supported by grants BFU2011–23604, SAF2014–52400-R, SAF2017–87846-R and S2013/ABI-2906 (PLATESA from Comunidad Autónoma de Madrid/ FEDER). Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged.

Antiviral agents are increasingly considered an option for veterinary medicine. An understanding of their mechanism of activity is important to plan their administration either as monotherapy or in combination with other agents. Previous studies have shown that the broad spectrum antiviral agent favipiravir (T-705) and its derivatives T-1105 and T-1106 are efficient inhibitors of foot-and-mouth disease virus (FMDV) replication in cell culture and in vivo. However, no mechanism for their activity against FMDV has been proposed. In the present study we show that favipiravir (T-705) can act as a lethal mutagen for FMDV in cell culture. Evidence includes virus extinction associated with increase in mutation frequency in the mutant spectrum of 860 residues of the 3D (polymerase)-coding region, and a decrease of specific infectivity while the consensus nucleotide sequence of the region analyzed remained invariant. The mutational spectrum evoked by favipiravir differs from that observed with other viruses in that no predominant transition type is observed, indicating that a movement towards A,U- or G,C-rich regions of sequence space is not a prerequisite for virus extinction. We discuss prospects for the use of favipiravir to assist in the control of FMDV, and its possible broader use in veterinary medicine as an extension of its current status as antiviral agent for human influenza virus.

Lethal mutagenesis is an antiviral approach that consists of extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagenic agent, often a nucleotide analogue. One of its advantages is its broad-spectrum nature, which renders the strategy potentially effective against emergent RNA viral infections. Here we describe the synergistic lethal mutagenesis of hepatitis C virus (HCV) by a combination of favipiravir (T-705) and ribavirin. Synergy has been documented over a broad range of analogue concentrations using the Chou-Talalay method implemented in CompuSyn graphics software, with the average dose reduction index (DRI) being above 1 (68.02 101.6 for favipiravir and 5.83 6.07 for ribavirin) and the average combination indices (CI) being below 1 (0.52 0.28). Furthermore, analogue concentrations that individually did not extinguish high-fitness HCV in 10 serial infections extinguished high-fitness HCV in 1 to 2 passages when used in combination. Although both analogues displayed a preference for G ¡ A and C ¡ U transitions, deep sequencing analysis of mutant spectra indicated a different preference of the two analogues for the mutation sites, thus unveiling a new possible synergy mechanism in lethal mutagenesis. The prospects for synergy among mutagenic nucleotides as a strategy to confront emerging viral infections are discussed, C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (grant CP14/00121), cofinanced by the European Regional Development Fund (ERDF). The Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) is funded by the Instituto de Salud Carlos III. The work in Madrid, Spain, was supported by grant SAF2014-52400-R from the Ministerio de Economía y Competitividad, grants SAF2017-87846-R, BFU2017-91384-EXP, and PI18/00210 from the Ministerio de Ciencia, Innovación y Universidades, grant S2013/ABI-2906 from PLATESA from the Comunidad de Madrid/FEDER, and grant P2018/BAA-4370 from PLATESA2 from the Comunidad de Madrid/FEDER. The work in Barcelona, Spain, was also funded by the Instituto de Salud Carlos III; by grant PI16/00337, cofinanced by the European Regional Development Fund (ERDF); and by the Centro para el Desarrollo Tecnológico Industrial (CDTI), Spanish Ministry of Economics and Competitiveness (MINECO) (grant IDI-20151125). Institutional grants from the Fundación Ramón Areces and Banco Santander to CBMSO are also acknowledged.

Rift Valley fever virus (RVFV) is an emerging, mosquito-borne, zoonotic pathogen with recurrent outbreaks taking a considerable toll in human deaths in many African countries, for which no effective treatment is available. In cell culture studies and with laboratory animal models, the nucleoside analogue favipiravir (T-705) has demonstrated great potential for the treatment of several seasonal, chronic, and emerging RNA virus infections in humans, suggesting applicability to control some viral outbreaks., Rift Valley fever virus (RVFV) is an emerging, mosquito-borne, zoonotic pathogen with recurrent outbreaks taking a considerable toll in human deaths in many African countries, for which no effective treatment is available. In cell culture studies and with laboratory animal models, the nucleoside analogue favipiravir (T-705) has demonstrated great potential for the treatment of several seasonal, chronic, and emerging RNA virus infections in humans, suggesting applicability to control some viral outbreaks. Treatment with favipiravir was shown to reduce the infectivity of Rift Valley fever virus both in cell cultures and in experimental animal models, but the mechanism of this protective effect is not understood. In this work, we show that favipiravir at concentrations well below the toxicity threshold estimated for cells is able to extinguish RVFV from infected cell cultures. Nucleotide sequence analysis has documented RVFV mutagenesis associated with virus extinction, with a significant increase in G to A and C to U transition frequencies and a decrease of specific infectivity, hallmarks of lethal mutagenesis.

The quasispecies dynamics of viral populations (continuous generation of variant genomes and competition among them) has as one of its frequent consequences variations in overall multiplication capacity, a major component of viral fitness. This parameter has multiple implications for viral pathogenesis and viral disease control, some of them unveiled thanks to deep sequencing of viral populations. Darwinian fitness is an old concept whose quantification dates back to the early developments of population genetics. It was later applied to viruses (mainly to RNA viruses) to quantify relative multiplication capacities of individual mutant clones or complex populations. The present article reviews the fitness concept and its relevance for the understanding of the adaptive dynamics of viruses in constant and changing environments. Many studies have addressed the fitness cost of escape mutations (to antibodies, cytotoxic T cells or inhibitors) as an influence on the efficacy of antiviral interventions. Here, we summarize the evidence that the basal fitness level can be a determinant of inhibitor resistance., Ministerio de Economía y Competitividad, SAF2017–87846-R, BFU2017–91384-EXP, PI18/00210 from Ministerio de Ciencia, Innovación y Universidades, S2013/ABI-2906, (PLATESA from Comunidad de Madrid/FEDER)

The influence of quasispecies dynamics on long-term virus diversification in nature is a largely unexplored question. Specifically, whether intra-host nucleotide and amino acid variation in quasispecies fit the variation observed in consensus sequences or data bank alignments is unknown. Genome conservation and dynamics simulations are used for the computational design of universal vaccines, therapeutic antibodies and pan-genomic antiviral agents. The expectation is that selection of escape mutants will be limited when mutations at conserved residues are required. This strategy assumes long-term (epidemiologically relevant) conservation but, critically, does not consider short-term (quasispecies-dictated) residue conservation. We calculated mutant frequencies of individual loci from mutant spectra of hepatitis C virus (HCV) populations passaged in cell culture and from infected patients. Nucleotide or amino acid conservation in consensus sequences of the same populations, or in the Los Alamos HCV data bank did not match residue conservation in mutant spectra. The results relativize the concept of sequence conservation in viral genetics and suggest that residue invariance in data banks is an insufficient basis for the design of universal viral ligands for clinical purposes. Our calculations suggest relaxed mutational restrictions during quasispecies dynamics, which may contribute to higher calculated short-term than long-term viral evolutionary rates. The research at Centro de Biologia Molecular Severo Ochoa (CSIC-UAM) (CBMSO) was funded by Ministerio de Economia y Competitividad (MINECO), grant numbers SAF2014-52400-R and SAF2017-87846-R, and by Ministerio de Ciencia, Innovacion y Universidades (MCIU), grant number BFU2017-91384-EXP, and by Instituto de Salud Carlos III, grant number PI18/00210, and by PLATESA from Comunidad de Madrid/FEDER, grant numbers S2013/ABI-2906 and S2018/BAA-4370. C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121 and CPII19/00001) cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigacion en Red de Enfermedades Hepaticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundacion Ramon Areces and Banco Santander to the CBMSO are also acknowledged. The research at Vall d'Hebron Institut de Recerca (VHIR) was funded by Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF), grant number PI19/00301 and by the Centro para el Desarrollo Tecnologico Industrial (CDTI) from the MCIU, grant number IDI-20151125. The research at Centro de Astrobiologia (CAB) was supported by MINECO, grant numbers BIO2016-79618R and PID2019-104903RB-I00 (funded by EU under the FEDER program) and by the Spanish State research agency (AEI), grant number MDM-2017-0737 (Unidad de Excelencia "Maria de Maeztu"-Centro de Astrobiologia (CSIC-INTA). C.G.-C. is supported by predoctoral contract PRE2018-083422 from MCIU. B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo) cofinanced by Fondo Social Europeo (FSE); With funding from the Spanish government through the "María de Maeztu Unit of Excellence" accreditation (MDM-2017-0737) Peer review

Viral RNA-dependent RNA polymerases (RdRps) are major determinants of high mutation rates and generation of mutant spectra that mediate RNA virus adaptability. The RdRp of the picornavirus foot-and-mouth disease virus (FMDV), termed 3D, is a multifunctional protein that includes a nuclear localization signal (NLS) in its N-terminal region. Previous studies documented that some amino acid substitutions within the NLS altered nucleotide recognition and enhanced the incorporation of the mutagenic purine analogue ribavirin in viral RNA, but the mutants tested were not viable and their response to lethal mutagenesis could not be studied. Here we demonstrate that NLS amino acid substitution M16A of FMDV serotype C does not affect infectious virus production but accelerates ribavirin-mediated virus extinction. The mutant 3D displays polymerase activity, RNA binding, and copying processivity that are similar to those of the wild-type enzyme but shows increased ribavirin-triphosphate incorporation. Crystal structures of the mutant 3D in the apo and RNA-bound forms reveal an expansion of the template entry channel due to the replacement of the bulky Met by Ala. This is a major difference with other 3D mutants with altered nucleotide analogue recognition. Remarkably, two distinct loop β9-α11 conformations distinguish 3Ds that exhibit higher or lower ribavirin incorporation than the wild-type enzyme. This difference identifies a specific molecular determinant of ribavirin sensitivity of FMDV. Comparison of several polymerase mutants indicates that different domains of the molecule can modify nucleotide recognition and response to lethal mutagenesis. The connection of this observation with current views on quasispecies adaptability is discussed., Work in Madrid was supported by grants BFU2011-23604, SAF2014-52400-R, and AGL2014-52395-C2-1-R from the Spanish MINECO and S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Work in Barcelona was supported by grants BIO2017-83906-P and Maria de Maeztu Unit of Excellence MDM-2014-0435, from the Spanish MINECO. X-ray data were collected at ALBA Synchrotron (Cerdanyola de Valles, Spain) XALOC beamline with the collaboration of ALBA staff and at ESRF beamline ID29 (Grenoble, France). Financial support was provided by ALBA and ESRF. The kinetic studies at the University of Missouri were partially supported by grant AI076119 (National Institutes of Health, USA) to S.G.S. C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/ 00121) cofinanced by the European Regional Development Fund (ERDF). Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged.

The selective pressures acting on viruses that replicate under enhanced mutation rates are largely unknown. Here, we describe resistance of foot-and-mouth disease virus to the mutagen 5-fluorouracil (FU) through a single polymerase substitution that prevents an excess of A to G and U to C transitions evoked by FU on the wild-type foot-and-mouth disease virus, while maintaining the same level of mutant spectrum complexity. The polymerase substitution inflicts upon the virus a fitness loss during replication in absence of FU but confers a fitness gain in presence of FU. The compensation of mutational bias was documented by in vitro nucleotide incorporation assays, and it was associated with structural modifications at the N-terminal region and motif B of the viral polymerase. Predictions of the effect of mutations that increase the frequency of G and C in the viral genome and encoded polymerase suggest multiple points in the virus life cycle where the mutational bias in favor of G and C may be detrimental. Application of predictive algorithms suggests adverse effects of the FU-directed mutational bias on protein stability. The results reinforce modulation of nucleotide incorporation as a lethal mutagenesis-escape mechanism (that permits eluding virus extinction despite replication in the presence of a mutagenic agent) and suggest that mutational bias can be a target of selection during virus replication, Work in Madrid was supported by grants [BFU2011-23604, SAF2014-52400-R, S2013/ABI2906 (PLATESA from Comunidad de Madrid/FEDER)] and Fundacion R. Areces and grant BFU2012-40020 to U.B. CIBERehd is funded by Instituto de Salud Carlos III. Work in Barcelona was supported by grants [BIO2011-24333, BIO2014-54588-P and MDM-2014-0435 from the Spanish MINECO]. X-ray data were collected at ALBA Synchrotron (Cerdanyola de Valles, Spain) XALOC beamline with the collaboration of ALBA staff. Financial support was provided by ALBA. C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121) cofinanced by the European Regional Development Fund (ERDF). The kinetic studies at the University of Missouri were partially supported by grant [AI076119] (National Institute of Health, USA) to S.G.S.

Low fidelity replication and the absence of error-repair activities in RNA viruses result in complex and adaptable ensembles of related genomes in the viral population, termed quasispecies, with important implications for natural infections. Theoretical predictions suggested that elevated replication error rates in RNA viruses might be near to a maximum compatible with viral viability. This fact encouraged the use of mutagenic nucleosides as a new antiviral strategy to induce viral extinction through increased replication error rates. Despite extensive evidence of lethal mutagenesis of RNA viruses by different mutagenic compounds, a detailed picture of the infectivity of individual genomes and its relationship with the mutations accumulated is lacking. Here, we report a molecular analysis of a foot-and-mouth disease virus population previously subjected to heavy mutagenesis to determine whether a correlation between increased mutagenesis and decreased fitness existed. Plaque-purified viruses isolated from a ribavirintreated quasispecies presented decreases of up to 200-fold in infectivity relative to clones in the reference population, associated with an overall eightfold increase in the mutation frequency. This observation suggests that individual infectious genomes of a quasispecies subjected to increased mutagenesis lose infectivity by their continuous mutagenic 'poisoning'. These results support the lethal defection model of virus extinction and the practical use of chemical mutagens as antiviral treatment. Even when extinction is not achieved, mutagenesis can decrease the infectivity of surviving virus, and facilitate their clearance by host immune responses or complementing antiviral approaches. © 2013 SGM., Ministerio de Ciencia e Innovación; Ministerio de Economía y Competitividad; Fundación Ramñon Areces; CIBERehd; CSIC; European Commission (Marie Curie)

Lethal mutagenesis is an antiviral approach that consists in extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagen. Here we show that favipiravir (T-705) is a potent mutagenic agent for hepatitis C virus (HCV) during its replication in human hepatoma cells. T-705 leads to an excess of G!A and C!U transitions in the mutant spectrum of preextinction HCV populations. Infectivity decreased significantly in the presence of concentrations of T-705 which are 2- to 8-fold lower than its cytotoxic concentration 50 (CC50). Passaging the virus five times in the presence of 400 μM T-705 resulted in virus extinction. Since T-705 has undergone advanced clinical trials for approval for human use, the results open a new approach based on lethal mutagenesis to treat hepatitis C virus infections. If proven effective for HCV in vivo, this new anti-HCV agent may be useful in patient groups that fail current therapeutic regimens., BFU-2011-23604, SAF2014-52400-R, S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER) and Fundación R. Areces. The work in Barcelona was funded by Instituto de Salud Carlos III, grants PI13-00456 and PI15-00829, cofinanced by the European Regional Development Fund (ERDF).

Lethal mutagenesis, a new antiviral strategy to extinguish virus through elevated mutation rates, was explored in H61-D cells an HIV-1 persistently infected lymphoid cell line. Three mutagenic agents: 5-hydroxy-2 '-deoxycytidine (5-OHdC), 5-fluorouracil (5-FU) and 2,2 '-difluoro-2 '-deoxycytidine (gemcitabine) were used. After 54 passages, treatments with 5-FU and gemcitabine reduced virus infectivity, p24 and RT activity. Treatment with the pyrimidine analog 5-OHdC resulted in increases of p24 production, RT activity and infectivity. Rise in viral replication by 5-OHdC during HIV-1 persistence is in contrast with its inhibitory effect in acute infections. Viral replication enhancement by 5-OHdC was associated with an increase in intracellular HIV-1 RNA mutations. Mechanisms of HIV-1 replication enhancement by 5-OHdC are unknown but some potential factors are discussed. Increase of HIV-1 replication by 5-OHdC cautions against the use, without previous analyses, of mutagenic nucleoside analogs for AIDS treatment. © 2011 Elsevier Inc., Fundacion para la Investigacion y Prevencion del SIDA en España (FIPSE); Red Temática Cooperativa de Investigación en SIDA (Red de grupos 173) of the Fondo de Investigaciones Sanitarias de la Seguridad Social; MICINN; Fundación Ramón Areces

Foot-and-mouth disease virus (FMDV) is one of the most contagious animal viruses, causing a devastating disease in clovenhoofed animals with enormous economic consequences. Identification of the different parameters involved in the immune response elicited against FMDV remains unclear, and it is fundamental the understanding of such parameters before effective control measures can be put in place. In the present study, we show that interleukin-10 (IL-10) production by dendritic cells (DCs) is drastically increased during acute infection with FMDV in swine. In vitro blockade of IL-10 with a neutralizing antibody against porcine IL-10 restores T cell activation by DCs. Additionally, we describe that FMDV infects DC precursors and interferes with DC maturation and antigen presentation capacity. Thus, we propose a new mechanism of virus immunity in which a non-persistent virus, FMDV, induces immunosuppression by an increment in the production of IL- 10, which in turn, reduces T cell function. This reduction of T cell activity may result in a more potent induction of neutralizing antibody responses, clearing the viral infection., This research was supported by grant AGL2004-0049, AGL2007-61374 and CSD2006-07 from Ministerio de Educacion y Ciencia, Spain and EU, Netwok of Excellence, EPIZONE (Contract # FOOD-CT-2006-016236). F.D.-S. was a fellowship of INIA. T.R. was supported by a contract from Comunidad Auto´noma de Madrid. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

In transmissible spongiform encephalopathies (TSEs) the prion protein (PrP) plays a central role in pathogenesis. The PrP gene (Prnp) has been described in a number of mammalian and avian species and its expression product, the cellular prion protein (PrPC), has been mapped in brains of different laboratory animals (rodent and non-human primates). However, mapping of PrPC expression in mammalian species suffering from natural (bovine and ovine) and experimental (swine) TSE or in species in which prion disease has never been reported (equine and canine) deserves further attention. Thus, localising the cellular prion protein (PrPC) distribution in brain may be noteworthy for the understanding of prion disease pathogenesis since lesions seem to be restricted to particular brain areas. In the present work, we analysed the distribution of PrPC expression among several brain structures of the above species. Our results suggest that the expression of PrPC, within the same species, differs depending on the brain structure studied, but no essential differences between the PrPC distribution patterns among the studied species could be established. Positive immunoreaction was found mainly in the neuropil and to a lesser extent in neuronal bodies which occasionally appeared strongly stained in discrete regions. Overall, the expression of PrPC in the brain was significantly higher in grey matter areas than in white matter, where accumulation of PrPSc is first observed in prion diseases. Therefore, other factors besides the level of expression of cellular PrP may account for the pathogenesis of TSEs. © Springer-Verlag 2006.

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious vesicular disease of cloven-hoofed animals. In the present study we use FMDV serotype C infection of swine to determine, by analytical techniques, the direct ex vivo visualization of virus-infected immune cells during the first 17 days of infection. We report, for the first time, that FMDV C-S8c1 can infect T and B cells at short periods of time postinoculation, corresponding with the peak of the viremia. There is a significant lymphopenia that involves CD3 + CD4 − CD8 +/− , CD3 + CD4 − CD8 + Tc, and CD3 + CD4 + CD8 + memory Th but not CD3 + CD4 + CD8 − naïve Th lymphocytes. In addition, a profound depletion of the vast majority of peripheral T cells in lymph nodes and spleen is observed. This selective depletion of T cells is not due mainly to in situ death via apoptosis as visualized by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique. Thus, early infection of T cells by FMDV may be the main cause of the observed T-cell depletion. Importantly, this lack of T cells is reflected in a reduced response to mitogen activation, which in many cases is totally eliminated. These data suggest a mechanism by which the virus causes a transient immunosuppression, subvert the immune systems, and spreads. These results have important implications for our understanding of early events in the development of a robust immune response against FMDV.

Foot-and-mouth disease virus (FMDV) is a picornavirus that causes an acute vesicular disease of cloven-hoofed animals. This virus continues to be threat to livestock worldwide with outbreaks causing severe economic losses. However, very little is known about FMDV pathogenesis, partially due to the inconveniences of working with cattle and swine, the main natural hosts of the virus. Here we demonstrate that C57BL/6 and BALB/C adult mice are highly susceptible to FMDV infection when the virus is administered subcutaneously or intraperitoneally. The first clinical signs are ruffled fur, apathy, humped posture, and wasting, which are followed by neurological signs such as hind-limb paralysis. Within 2-3 days of disease onset, the animals die. Virus is found in all major organs, indicating a systemic infection. Mice developed microvesicles near the basal layer of the epithelium, event that precedes the vesiculation characteristics of FMD. In addition, a lymphoid depletion in spleen and thymus and severe lymphopenia is observed in the infected mice. When these mice were immunized with conventional inactivated FMDV vaccine, they were protected (100% of vaccinated animals) against challenge with a lethal dose of FMDV. The data indicate that this mouse model may facilitate the study of FMDV pathogenesis, and the development of new effective vaccines for FMD. © 2004 Elsevier Inc. All rights reserved.